Epac1 Signaling Pathway Mediates The Damage And Apoptosis Of Inner Ear Hair Cells After Noise Exposure In A Rat Model

Objective:To investigate the role of the exchange protein directly activated by c AMP（Epac）signaling pathway in inner ear hair cell damage and apoptosis after noise exposure,we analyzed the expression levels of Epac1 in a rat model of noise-induced hearing loss（NIHL）.Methods:Forty SD rats were randomly divided into normal control group,noise exposuregroup（NE）,NE+8-CPT and NE+ESI-09.The rats of noise exposure were exposed to4 k Hz at 106 d B sound pressure level（SPL）for 8 h.Auditory brainstem responses（ABR）were measured in rats before noise exposure and 24 h after noise exposure.Surface preparation,Transmission electron microscopy（TEM）and Immunohistochemical were performed on cochlea tissues,to elucidate changes in Epac expression in rat after noise exposure.The expression levels of Epac1、Rap1、CaMKII、Bax、Bcl-2 and Cleaved caspase3 were analyzed using western blot.Moreover,the ATP content in the cochlea after noise exposure was detected by an ATP detection kit.A previous study has shown that noise induces a transient ATP depletion in the cochlear tissue,which results in cochlear hair cells death.Oligomycin,an ATP synthase inhibitor,activates Epac1 and was used in this study to induce damage in HEI-OC1 cells similar to that in cochlear hair cells following noise exposure in vitro.In the HEI-OC1 cell model,the optimal concentration for oligomycin-induced HEI-OC1 cell damage was detected by CCK-8.The morphological and proliferation-related changes of HEI-OC1 cells were observed by holographic microscope imaging after oligomycin exposure.Cell apoptosis was determined by using Annexin-V FITC/PI staining.The expression levels of Epac1、Rap1、CaMKII、Bax、Bcl-2 and cleaved caspase3 were analyzed using western blot.The effects of 8-CPT and ESI-09 on Ca²⁺entry were evaluated using confocal Ca²⁺fluorescence measurement.Results:Compared with Control group,the ABR threshold of the NE group was significantly increased.Compared with NE group,the agonist of Epac increased ABR threshold,but there was no difference.The inhibitor of Epac decreased the rise of the threshold.Surface Preparations results show that the three layers of outer hair cells were neatly arranged,without deletion in control group.The missing of outer hair cells occurred in NE group and NE+8-CPT group.Compared with NE group,NE+ESI-09 group decreased hair cell loss.Transmission electron microscopy（TEM）further proved the above changes and show that outer mitochondrial membrane rupture,cristae disappearance and vacuolation in NE group.The agonist of Epac increased mitochondrial damage.The inhibitor of Epac decreased mitochondrial ultrastructure damage in the cochlea of rats.Immunofluorescence and Western blot show that the level of Epac1 were significantly higer than the Con group,the Epac agonist increased the level of Epac1,the Epac inhibitor significantly decreased the level of Epac1.Meantime,the levels of CaMKII,Rap1 and apoptotic protein Bax,cleaved caspase3 significantly increased,and the level of Bcl-2 decreased in the NE group.The Epac agonist further increased the level of CaMKII,Rap1 and apoptotic protein Bax,cleaved caspase3,and promote hair cell apoptosis.The Epac inhibitor significantly decreased the CaMKII,Rap1 and apoptotic protein Bax,cleaved caspase3,and Inhibits hair cell apoptosis after noise exposure.Oligomycin was used to induce injury in HEI-OC1 cells in vitro.The results of CCK-8 showed that the optimal concentration for oligomycin-induced damage in HEI-OC1 cells was 1μM.Holographic microimaging showed reduced cell confluency in oligomycin-treated（OA）cells compared to controls,with further reduction in cell confluency by the Epac agonist 8-CPT.Epac inhibitor ESI-09 increased cell confluency compared with the OA group.Immunofluorescence showed the level of Epac1 were significantly higer than the Con group,the Epac agonist increased the level of Epac1,the Epac inhibitor significantly decreased the level of Epac1.Annexin-V FITC/PI staining showed that compared with the Control group,the apoptosis of HEI-OC1 cells in the OA group was increased.Epac agonist 8-CPT aggravated the apoptosis of HEI-OC1 cells,but the inhibitor ESI-09 could reduce the apoptosis of HEI-OC1 cells.Western blot results showed that in OA group,the expressions of Rap 1 and CaMKII proteins in HEI-OC1cells were increased,the expression of apoptotic protein Bax was up-regulated.Moreover,oligomycin could induce the activation of caspase3 in HEI-OC1 cells,and the expression of cleaved caspase3 was significantly increased.Epac agonist 8-CPT further promoted the expression of the above proteins and promoted the apoptosis.Epac inhibitor ESI-09 could significantly inhibit the expression of Rap 1 and CaMKII proteins in HEI-OC1 cells,and inhibit oligomycin-induced apoptosis.Moreover,8-CPT promoted Ca²⁺uptake in HEI-OC1 cells,while ESI-09 inhibited this process.Conclusion:In conclusion,the results provide strong evidence that the Epac1 signaling pathway mediates early pathological damage in NIHL,and that Epac1 inhibition protects against NIHL,thereby suggesting Epac1 as a new potential therapeutic target for NIHL.